Journal: Plant biology (Stuttgart, Germany)

Article Title: The transmembrane domain of the rice small protein OsS1Fa1 is responsible for subcellular localization and drought tolerance.

doi: 10.1111/plb.13711

Figure Lengend Snippet: Fig. 2. Analysis of OsS1Fa1 and OsS1Fa2 expression in rice tissues and detection of the His-tagged recombinant proteins in E. coli. (A) A comparison of the deduced amino acid sequences of OsS1Fa1 and OsS1Fa2. (B) Amino acid sequences of conserved motifs in OsS1Fa1 and OsS1Fa2 are indicated. (C) Total RNA was extracted from leaf, culm, and root tissues of 3-week-old rice seedlings, and the expression levels of OsS1Fa1 and OsS1Fa2 were analysed using qRT-PCR. Data represent mean SD (n = 3). (D) Detection of recombinant His6-OsS1Fa1 and His6-OsS1Fa2 proteins with anti-OsS1Fa1 antibody.

Article Snippet: Rice leaf, culm, and root samples were thoroughly ground, and total RNA was extracted from the ground tissue using the FavorPrepTM Plant Total RNA Mini Kit (Favorgen).

Techniques: Expressing, Recombinant, Comparison, Quantitative RT-PCR

Journal: Plant biology (Stuttgart, Germany)

Article Title: The transmembrane domain of the rice small protein OsS1Fa1 is responsible for subcellular localization and drought tolerance.

doi: 10.1111/plb.13711

Figure Lengend Snippet: Fig. 4. Drought tolerance assay of transgenic Arabidopsis overexpressing OsS1Fa1 and mutant proteins. (A) Examination of OsS1Fa1, OsS1Fa1(TMm1), OsS1Fa1(mNLS ), and OsS1Fa1(K63R) expression in OsS1Fa1-, OsS1Fa1(mNLS)-, OsS1Fa1(K63R)-, and OsS1Fa1(TMm1)-overexpressing Arabidopsis. Total RNA was extracted from the leaves of 15-day-old WT and transgenic Arabidopsis, and then OsS1Fa1, OsS1Fa1(TMm1), OsS1Fa1(mNLS ), OsS1Fa1(K63R) transcripts were examined by RT-PCR using OsS1Fa1- and FLAG-specific primers. (B) Seeds of WT, OsS1Fa1-, OsS1Fa1(mNLS)-, OsS1Fa1(K63R)-, and OsS1Fa1(TMm1)- overexpressing Arabidopsis were grown for 15 days in moist soil, and watering was withheld for 7 days for drought stress treatment. Following the drought stress treatment, the plants were rewatered, and photographs taken after an additional 3 and 5 days.

Article Snippet: Rice leaf, culm, and root samples were thoroughly ground, and total RNA was extracted from the ground tissue using the FavorPrepTM Plant Total RNA Mini Kit (Favorgen).

Techniques: Transgenic Assay, Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: iScience

Article Title: An AI-driven workflow for the accelerated optimization of cell-free protein synthesis

doi: 10.1016/j.isci.2025.113599

Figure Lengend Snippet: Detecting and quantifying cell-free synthesized proteins (A) Split-GFP tag consists of GFP α-helix 11 as a tag fused to any of the colicins. Co-expression of truncated GFP1-10 and GFP11 tagged with colicins in a transcription-translation (TX-TL) coupled CFPS system restores fluorescent GFP. (B) Split-Nanoluciferase consists of the HiBiT domain as a tag fused to the colicin. After the TX-TL CFPS reaction, the recombinant large subunit of the luciferase (LgBiT) is added together with luciferase substrate to assemble a functional Nanoluciferase. (C) Kinetics of fluorescence detection after the co-expression of the gene encoding GFP1-10 and GFP11 with colicins (dark green), or single expression of GFP1-10 (coral) or GFP11 (purple). Positive controls were sfGFP (dark red) and the combined GFP1-10 and GFP11 (blue). Both assays were performed in 10.5 μL reactions at 30°C, with all the fluorescence readings normalized to the No-DNA control. All plasmid DNA final concentrations were 6 nM for the split-GFP-ColM experiment and all DNA were 5 nM for the split-GFP-ColE1 experiment. (D) Data are presented as means ± SD for six independent experiments ( n = 6) using a unique batch of recombinant his-HiBit-GFP purified proteins. Design and purification of the His-HiBiT-GFP1-11 to compare fluorescence and luminescence sensitivity thresholds. The protein was purified using the His tag and quantified using the Bradford method. A range from 1 to 10 ng of recombinant protein was detected by Western blot (top panel) using His antibody (α-His), GFP antibody (α-GFP) or HiBiT antibody (α-HiBiT). The untrimmed gel is shown in . A range of 1–10 ng then 0.1 to 1 ng of His-HiBiT-GFP1-11 was quantified by fluorescence (green) and luminescence (orange). Readings were measured from the same well deposit with 6 replicates in a final volume of 2 μL for fluorescence, then 2 μL of substrate and LgBiT were added for luminescence. The Pearson correlation coefficient ( R 2 ) was used to determine the sensitivity threshold for both fluorescence and luminescence.

Article Snippet: The plasmids were isolated from a 300 mL LB of E. coli DH5α using the Plasmid DNA purification NucleoBond Xtra Maxi of Macherey-Nagel.

Techniques: Synthesized, Expressing, Recombinant, Luciferase, Functional Assay, Fluorescence, Control, Plasmid Preparation, Purification, Western Blot